7-(3-substituted-1,2,4-oxadiazole-5-one-4-acetamido)cephalosporanic acids and derivatives thereof

ABSTRACT

6 - (N (SUBSTITUTED - IMIDOLYL)AMINOACETAMIDO)CEPHALOSPORANIC ACIDS AND THEIR SALTS AND THE CORRESPONDING BETAINES AND DESACETOXY DERIVATIVES ARE VALUABLE AS ANTIBACTERIAL AGENTS, IN POULTRY AND ANIMALS, INCLUDING MAN, THERAPEUTIC AGENTS IN POULTRY AND ANIMALS, INCLUDING MAN, AND ARE ESPECIALLY USEFUL IN THE TREATMENT OF INFECTIOUS DISEASES CAUSED BY GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA. 6 - (N - PHENYLACETIMIDOYL)AMINOACETAMIDO)CEPHALOSPORANIC ACID, A PREFERRRED EMBODIMENT OF THE INVENTION, IS PREPARED FOR EXAMPLE, BY RANEY NICKEL HYDROGENATION (50 P.S.I., R.T.) OF AN AQUEOUS SOLUTION OF SODIUM 7 - (3 - BENZYL - 1,2,4 - OXADIAZOLE - 5 - ONE - 4 - ACETAMIDO)CEPHALOSPORANATE WHICH IS PREPARED IN TURN BY REACTION OF 7 - AMINOCEPHALOSPORANIC ACID WITH 3 - BENZYL1,2,4-OXADIAZOLE-5-ONE-4-ACETYL CHLORIDE.

' States Patent O 3,833,570 7-(3-SUBS'I'ITUTED 1,2,4 OXADIAZOLE-S-ONE-4-ACETAMIDO)CEPHALOSPORANIC ACIDS AND DERIVATIVES THEREOF Charles TrumanHoldrege, Camillus, N.Y., assignor to Bristol-Myers Company, New York,N.Y. No Drawing. Original application Aug. 12, 1970, Ser. No. 63,314,now Patent No. 3,692,779, dated Sept. 19, 1972. Divided and thisapplication Aug. 31, 1972, Ser.

Int. Cl. C0711 99/24 U.S. Cl. 260243 C 12 Claims ABSTRACT OF THEDISCLOSURE 6 [N (substituted imidoyl)aminoacetamido]cephalosporanicacids and their salts and the corresponding betaines and desacetoxyderivatives are valuable as antibacterial agents, nutritionalsupplemcnts in animal feeds, therapeutic agents in poultry and animals,including man, and are especially useful in the treatment of infectiousdiseases caused by Gram-positive and Gram-negative bacteria. 6 [NPhenylacetimidoyl)aminoacetamido]cephalosporanic acid, a preferredembodiment of the invention, is prepared for example, by -Raney nickelhydrogenation (50 p.s.i., RT.) of an aqueous solution of sodium 7 (3benzyl 1,2,4 oxadiazole one 4 acetamido)cephalosporanate which isprepared in turn by reaction of 7 aminocephalosporanic acid with 3benzyl- 1,2,4-oxadiazole-5-one-4-acetyl chloride.

application Ser. No. 63,314, filed Aug. 12, 1970 and issued Sept. 19,1972 as U.S. Pat. 3,692,779.

BACKGROUND OF THE INVENTION (1) Field of the Invention Thecephalosporins of the present invention possess the usual attributes ofsuch compounds and are particularly useful in the treatment of bacterialinfections.

(2) Description of the Prior Art Cephalothin and cephaloridine arewell-known antibacterial agents; see U.S. Pats. 3,218,318; 3,449,338 and3,498,979. The literature also contains considerable data on theactivity of cephaloglycin and cephalexin; see U.S. Pats. 3,303,193 and3,507,861 and Great Britain 985,747 and 1,054,806. Newer cephalosporinsinclude cefazolin and cephapirin; see U.S. Pat. 3,516,997 [and alsoNethererlands 680.5179 (Farmdoc 34,328) and South Africa 68/4,513] andU.S. Pat. 3,422,100.

The literature on cephalosporins has been reviewed by E. P. Abraham,Quart. Rev. (London) 21, 231 (1967), by E. Van Heyningen, Advan. DrugRes. 4, 1-70 (1967) and briefly in Annual Reports in MedicinalChemistry, Academic Press, Inc., 111 5th Ave., New York, N.Y. 10003, byL. C. Cheney on pages 96 and 97 (1967 and by K. Gerzon and R. B. Morinon pages 90-93 (1968). New cephalosporins are frequently reported at theannual Interscience Conference on Antimicrobial Agents and Chemotherapyas illustrated by Sassiver et al., Antimicrobial Agents andChemotherapy1968, American Society for Microbiology, Bethesda, Md.,pages 101-114 (1969) and by Nishida et al., ibid., 236-243 (1970).

7 (a Aminoacetamido)cephalosporanic acid is disclosed in U.S. Pat.3,422,103 and 7 (guanidinoacetamido)cephalosporanic acid in U.S. Pat.3,492,297.

With regard to the cephalosporins used an intermediates in the presentinvention, numerous methylcephalosporins,

lcei i.e., 7-acetamidocephalosporanic acids, having attached at thealpha position a heterocyclic ring containing two or three or fourhetero-atoms and often other substituents on the heterocyclic nucleusare reported in the literature, e.g., in U.S. Patents classified insub-class 260243 as exemplified most recently by U.S. Pats. 3,499,893;3,468,- 874; 3,459,746; 3,464,999; 3,365,449; 3,360,515 and 3,351,597.

The preparation of various 7-[a-amino-arylacetamido] cephalosporanicacids and the corresponding desacetoxy compounds in which arylrepresents unsubstituted or substituted phenyl or 2- or 3-thienyl isdescribed in British Specifications 985,747, 1,017,624, 1,054,806 and1,123,333, in Belgian Pat. 696,026 (Farmdoc No. 29,494), in U.S. Pats.3,311,621 and 3,352,858, in Japanese Pat. 16,871/66 (Farmdoc 23,231), bySpencer et al., J. Med. Chem., 9(5), 746-750 (1966) and by Kurita etal., J. Antibiotics (Tokyo) (A) 19, 243-249 1966) and see also U.S. Pat.3,485,819.

Netherlands Pats. 6811676 (Farmdoc 36,349) and 6812382 (Farmdoc 36,496)and U.S. Pats. 3,489,750 and 3,489,751 disclose ring-substitutedcephaloglycins.

Various 7 [a amino arylacetamido] cephalosporins in which one hydrogenof the a-amino group is replaced by a carbonyl group which is attachedin turn to another moiety have been reported. The earliest were thecephaloglycin and cephalexin precursors in which use was made of acommon peptide blocking group such as carbobenzyloxy as illustrated byU.S. Pat. 3,364,212. Belgian Pat. 675298 (Farmdoc 22,206), South AfricanPat. 67/1,260 (Farmdoc 28,654) and Belgian Pat. 696,026 (Farmdoc29,494). Later examples include my own7-(a-3-acylureido-arylacetamido)cephalosporins in my U.S. applicationSer. No. 792,757, filed Jan. 21, 1969, and issued Aug. 29, 1972 as U.S.Pat. 3,687,949 and my own 6-[m- (3imidoylureido)arylacetamido]cephalosporins in my U.S. application Ser.No. 855,375, filed Sept. 4, 1969, and the 7 [a (3 guanyl 1ureido)arylacetamido1cephalosporins of my colleague, Donald NeilMcGregor, disclosed in his U.S. application 793,909, filed J an. 24,1969 and issued May 18, 1971, as U.S. Pat. 5,579,514.

The penicillins having the same side-chains as the cephalosporins of thepresent invention are the subject of my U.S. application Ser. No.50,997, filed June 29, 1970 and issued June 13, 1972 as U.S. Pat.3,669,958 in U.S. Class 260/239.1 and my U.S. application Ser. No. 203,-042, filed Nov. 29, 1971 and issued Oct. 23, 1973 as U.S. Pat.3,767,646.

SUMMARY OF THE INVENTION Compounds having the formulae wherein A ishydrogen, acetoxy, 2-(5-methyl-l,3,4-thiadiazolyl)thio, pyridinium,picolinium or lutidinium; M is hydrogen or a pharmaceutically acceptablenontoxic cation or an anionic charge when A is one of said quaternaryammonium radicals; and wherein Z is (lower)alkyl, cycloalkyl having 4,5, 6 or 7 carbon atoms, m0nohalo(lower) wherein n is zero or one and R Rand R each is hydrogen, nitro, di(lower)alkylamino,(lower)alkanoylamino, (lower)alkanoyloxy, (lower-)alkyl (comprisingstraight and branched chain saturated aliphatic groups having from 1 to6 carbon atoms inclusive), amino, hydroxy, (lower) alkylthio,(lower)alkoxy, sulfamyl, chloro, iodo, bromo, fluoro or trifluoromethyl.Typical nontoxic, pharmaceutically acceptable cations include metalliccations such as sodium, potassium, calcium, aluminum, and the like, theammonium cation and substituted ammonium cations, e.g., cations of suchnontoxic amines as tri(lower)alkylamines, i.e., triethylamine, etc.,procaine, dibenzylamine, N-benzyl-beta-phenethylamine, l-ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine,N,N-bis-dehydroabietylethylenediamine, N (lower)alkylpiperidine, e.g.,N-ethylpiperidine, or other such amines which have been used to formpharmaceutically acceptable nontoxic salts with benzylpenicillin. Suchsalts are perferably made by treatment of the free acid (zwitterion inthe compounds of formula I) form of the product with a strong base. Inaddition the compounds of formula I form acid addition salts includingthe mineral acid addition salts such as the hydrochloride, hydrobromide,hydroiodide, sulfate, sulfamate and phosphate and the organic acidaddition salts such as the maleate, fumarate, malate, mandelate,ascorbate, para-toluenesulfonate, B-naphthalenesulfonate', and the like.

In the preferred embodiments of this invention the compounds exist inthe zwitterion form and Z is phenyl, benzyl, thenyl, thienyl, furyl or(1ower)alkyl and preferably benzyl, Z-thenyl or 3-thenyl.

The compounds of Formula II above are converted into those of Formula Iby hydrogenation and preferably by hydrogenation at about roomtemperature over Raney nickel catalyst of an aqueous solution of a saltor betaine of the compounds of Formula II and preferably a sodium orpotassium salt. The hydrogen pressure is preferably about 50 p.s.i. orhigher.

The betaines of Formula H are produced by reacting the compounds abovein which A is acetoxy with pyridine, picoline or lutidine by the methodof Spencer et al., J. Org. Chem., 32, 500 (1967).

The 3 [2 (S-methyl-1,3,4-thiadiazolyl)thiomethyl]- compounds areproduced by reacting a compound above in which A is acetoxy with halfits weight of S-methyl- Z-mercapto-1,3,4-thiadiazole, preferably in anaqueous system, e.g., in phosphate buffer at pH 6.4 for a few hours at60 C. The desired product precipitates as an oil or a solid.

The compounds of Formula II above can be prepared by direct acylation of7-aminocephalosporanic acid or 7-amino-3-desacetoxycephalosporanic acidor a salt thereof with an acid chloride of the formula (wherein Z hasthe meaning set out above) or its functional equivalent as an acylatingagent for a primary amino group using the conditions 'known to the artfor the preparation of compounds such as cephalothin and those describedin US. Pat. 3,322,750, for example.

DETAILED DESCRIPTION It was an object of the present invention toprovide compounds useful in the treatment of infections caused byGram-positive and Gram-negative bacteria, including particularly thosecaused by enteric organisms.

The compounds of the present invention as defined above under Summary ofthe Invention are particularly useful in that they possess potentantibacterial activity against both Gram-positive and Gram-negativebacteria, and most particularly those having structure I exhibit verypotent activity in vitro against enteric organisms which are resistantto the commercially available cephalosporins.

The compounds of the present invention containing thea-(3-substituted-1,2,4-oxadiazole-5-one-4-yl) group are primarily usefulas intermediates for the preparation of the corresponding compoundscontaining the 7-N-(substituted-imidoyl)aminomethyl side chain. Thelatter are far more potent antibacterial agents.

In the treatment of bacterial infections in man, the compounds of thisinvention containing the latter side chain are administered topically,orally and especially parenterally in accordance with conventionalprocedures for antibiotic administration in an amount of from about 5 to125 mg./kg./day and preferably in the range of 15 to 50 mg./kg./day individed dosages, e.g., three or four times a day. They are administeredin dosage units containing, for example, 125, 250, 500, 1000 and 2000mg. of active ingredient with suitable physiologically acceptablecarriers or excipients. The dosage units can be in the form of liquidpreparations such as solutions, dispersions, emulsions or in solid formsuch as tablets, capsules, etc.

The compounds of the present invention are also useful fordecontamination of filling machines and containers used for cosmeticsand topical drugs to prevent or at least reduce the very commoncontamination of such products with Gram-negative microorganisms. Insuch instance, use is made of an aqueous solution having a concentrationof at least 1 mgm./ml and a contact time of at least one hour.

The starting acids of the formula are prepared by standard methods ofsynthesis from known nitriles by the following reaction scheme wherein Zhas the meaning set out above (and as illustrated in the Examplesbelow):

NazO 03 NOH H2O-EtOH 7 8 TABLE-Continued TAB'LEContinued StructureReferences Structure References a n tg;t%.1gt3ltit N f E cited therein.l ALTERNATIVE METHOD FOR PREPARING COM- POUNDS OF GENERAL FORJM'ULA IICN i-iifiif available (Ald- 10 Compounds of general formula II can beprepared I by reaction of 7-bromoacetamidocephalosporanic acid \N with a3-substituted-1,2,4-oxadiazole-5-one as follows:

S z NH BrzCHzCONH- I l t \N 0N =o O=N C -omo-o-orn (IJOOH Aromatic andaliphatic nitriles are well-known (EJI'J- B selrEAfi aifiqutidine poundswhich are prepared, for example, by the met 0 s lsolvent-T ioxane.outlined in classic textbooks such as Organic Preparations CHZCI" CHCh'or DMF by Conrad Weygand, Interscience Publishers, Inc., New Z 3 York(1945) on pages 115, 255 and 367 and The Chemistry of the CarbonCompounds by Victor Van Richter N 0 N CH edited by Richard Anschutz,Third English Edition, El- \0/ f 1 3 sevier Publishing Co., Inc., NewYork, N.Y. (1946) in II (1300B Volume III at pages 304-306 and in VolumeI at pages 325327 and Laboratory Methods of Organic Chemistry5-Hydroxy-3-(2'-thienyl)-1,2,4-oxadiazole.A mixture by L. Gattermannrevised by H. Wieland, translated from of 176.1 g. (1.61 mole) of2-thienonitrile, 11.9 g. (1.61 the twenty-fourth edition, MacMillan andCo., Limited, mole) of hydroxylamine hydrochloride, 85.3 g. (0.805London (1943) at pages 137-138 and by the following mole) of sodiumcarbonate, 1 liter of 95% ethanol and procedure: 300 ml. of water washeated at reflux for 17 hours. A

2-Furamide (11.1 g., 0.1 mole, Eastman Kodak Co.) small amount of solidwas removed by filtration. Addiand phosphorus pentoxide (21 0.15 mole)were mix d tional water was added to the filtrate and the ethanol andheated with a Meeker burner (gently at first and then Was pp Off atIfidllced Pressure causing the Ystal more strongly) until 2-cyanof-urandistilled at l30-140 line product to separate. The filtered product waswashed C., 5.6 g. with water. The crude product was heated with benzeneSuch amides are prepared by published m thod as ilmost of it going intosolution. Cooling this mixture gave lustrated in the following table inwhich it is to be underafter filtration and drying 170.9 g. of2-thiophenecarstood that acids are converted to acid chlorides bytreatboxamide OXiIne; -P- 87-88O Beilment with thionyl chloride and acidchlorides are cony chlolofofmate 111016) added verted to amides byreaction with ammonia: gradually to a heated and stirred solution of170.9 g. (1.2 mole) of 2-thiophenecarboxamide oxime, 96.5 ml. AmideIllustrative Sources of pyrldme and 300 ml. of benzene. An exothermicreaction took place which caused the mixture to reflux. I F w} ggl fgg'fg gfi gizkf g aa The mixture was refluxed for 15 minutes after theaddis N A8351? 13% chloride ethyl ester tron of1etclllylrctllilloroformate was complete. The mixture Huird M011, Am"Chm Sam L135 coo e s 1g t y and 600 m1. of. water was added. The

5362 (1955). enzene was distilled off and the remaining aqueous ITllX-CONH2 Acid ethyl ester, amide: ture was refluxed for 2.5 hours. Aftercooling overnight H the product was filtered and washed with water. TheN\ /N crude product was dissolved in dilute aqueous sodium S hydroxide,the solution carbon treated, and reprecipitated CH3 1 CONH: ficidaethglzsttig gzzwigla. tk by acidification with acetic acid. There wasobtained N J: g a 'Lu-bosfmikovh, f 8 139.4 g. of5-hydroxy-3-(2'-thienyl)-1,2,4-oxadiazole; m .p. \0 CH Khim.32,177885(1962). 185-190 dec. The nuclear magnetic resonance and in- I I t fraredspectra were consistent for the desired compound. ggg f fgl 72 6O3-(2'-Furyl)-5-hydroxy-l,2,4-oxadiazole. A mixture N y Quilieo, StagnodAleontres, Gazz. 0f 69.5 g. (0.746 mole) of 2-furylnitrile [Ber. 14,1058 \O OONHz g ggi fg? Soc 1881], 51.8 g. (0.746 mole) of hydroxylaminehydro- 1962,4234 chloride, 39.5 g. (0.373 mole) of sodium carbonate, NAmidemommercially available; 500 ml. of 95% ethanol and 150 ml.'of waterwas re- OONHZ fluxed overnight. The mixture was filtered and 150 ml. ofwater was added. The ethanol was stripped 011 at \N reduceld pressure}.1Tfie1 aqueous phase was extracted h I severa times Wit et y acetate. Thecombined organic IF fi 3 3 33? ,ffl fiq m by extracts were carbontreated, dried with sodium sulfate N N known methods. and the solventremoved at reduced pressure leaving 88.9 g. of 2-furylamide oxime as aviscous oil (lit. ref. C.A.

Acid, acid chloride: Doyle and Nay- 7292b) CONHZ 1 U.S. 2,996,501 andreferences Ethyl chloroformate (76.5 g., 0.705 mole) was added Ii J: medtheremslowly to a heated and stirred solution of 88.9 g. of \0 CH3Z-furylamrde oxime, 56.7 ml. (0.705 mole) of pyridine and 200 ml. ofbenzene. After the addition of ethyl chloroformate was complete thereaction mixture was refluxed an additional 15 minutes. Water (400 ml.)was added and the benzene distilled off. The remaining aqueous mixturewas refluxed for 2.5-3 hours. After cooling in an ice bath the solidproduct was collected by filtration and washed with water. The productwas dissolved in dilute aqueous sodium hydroxide solution, carbontreated, the solution acidified with acetic acid with ice cooling andthe reprecipitated product filtered and washed carefully with ice water.The product was again purified in the same manner giving after drying invacuo over phosphorus pentoxide 50.1 g. of 3-(2-furyl)-5-hydroxy-1,2,4-oxadiazole; dec. above 210.

Analysis.Calcd. for C H N O C, 47.37; H, 2.65; N, 18.42. Found: C,47.27; H, 2.91; N, 18.39.

Illustrative examples of the preparation of compounds of the presentinvention follow. These examples are given in illustration of, but notin limitation of, the present invention. All temperatures are in degreesCentigrade. Skellysolve B is a petroleum ether fraction of b.p. 6068 C.consisting essentially of n-hexane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1S-Benzyl-1,2,4-oxadiazo1e-5-0ne.A solution of 117.2 g. (1 mole) ofphenylacetonitrile in 200 ml. 95% ethanol was added to a stirred mixtureof 69.5 g. (1 mole) of hydroxylamine hydrochloride, 53 g. (0.5 mole) ofsodium carbonate, 200 ml. of water and 400 ml. of 95% ethanol. Themixture was heated at a reflux for 3.5 hours, stored at room temperaturefor two days then refluxed for 3 hours longer. The ethanol was distilledoff at reduced pressure. Benzene was added to the residue and theaqueous layer was removed. The benzene solution containingphenylacetamide oxime was dried with sodium sulfate, filtered, andconcentrated to a volume of about 400 ml. Reference to phenylacetamideoxime: Beil. 9, 446, II 305.

Ethyl chloroformate (9 6 ml., 1 mole) was added gradually from adropping funnel to the above prepared solution of phenylacetamide oximein benzene plus 80.5 ml. (1 mole) of pyridine initially at roomtemperature. The exothermic reaction caused the mixture to reflux. Themixture was refluxed for 15 minutes longer, cooled somewhat and 500 ml.of water added. The benzene was distilled off and the remaining aqueousmixture was heated at reflux for 2.5 hours longer. The oil whichseparated was extracted into benzene. The benzene phase was extractedwith 175 ml. of 20% sodium hydroxide solution and then with 50 ml. ofwater. The combined basic extracts were acidified with acetic acidgiving oily crystals. The crude product was recrystallized from benzene;m.p. 80-94", yield 41 g. The product was combined with 100 ml. of waterand 50 ml. of 20% sodium hydroxide solution and the mixture heated forone hour on the steam bath. The cooled solution was acidified withacetic acid. An additional 100 ml. of water was added to make themixture more fluid. The product was collected by filtration. The dampfilter cake was dissolved in about 500 ml. of chloroform with warming.The solution was dried with sodium sulfate, filtered, and the solventdistilled off at reduced pressure. The residue was extracted with three250 ml. portions of boiling cyclohexane and with 500 ml. of boiling 1:1cyclohexanebenzene. The insoluble residue was slurried with cyclohexaneand collected by filtration. There was obtained 25.2 g. of3-benzyl-1,2,4-oxadiazole-5-one; m.p. l09-l12 (Beil. 27, II 710 reportsm.p. 115). The infrared spectrum was consistent for the desired product.

3-benzyl-1,2,4-oxadiazole-5-one-4-acetic acid.A solution of 25 g. (0.142mole) 3-benzyl-1,2,4-oxadiazole-5- one in 300 ml. of methanol wasstirred with 20 g. of pow dered Molecular Sieves (Linde 4A) for one hourat room temperature. The Molecular Sieves were removed by filtration. Tothe stirred filtrate was added 8.45 g. (0.156 mole) of sodium methoxideand then 26.2 g. (0.156 mole) of ethyl bromoacetate. The mixture washeated at reflux for 18 hours. The solvent was distilled off at reducedpressure. To the residue was added 200 ml. of acetic acid and ml. of 6 Nhydrochloric acid and the mixture refluxed for 1.5 hours. The solventswere distilled off at reduced pressure. To the residue was added waterand benzene causing the product to crystallize. The product wasfiltered, washed on the filter with water and benzene and air dried. Thecrude product was recrystallized from toluene with a carbon treatmentgiving 15.9 g. of white crystalline product after drying in vacuo overphosphorus pentoxide; m.p. 111113. The infrared and nuclear magneticresonance spectra were consistent for the desired product.

Analysis.-Calcd. for C H N O C, 56.41; H, 4.30; N, 11.96. Found: C,56.61; H, 4.42; N, 12.21.

3 Benzyl-1,2,4-oxadiazole-5-one-4-acetyl chloride.-A solution of 5.0 g.(0.0214 mole) of 3-benzyl-1,2,4-oxadiazole-5-on-4-acetic acid in 200 ml.of methylene chloride was gassed briefly (2 minutes) with hydrogenchloride. To the resulting clear solution was added 5.8 g. (0.0278 mole)of phosphorus pentachloride (effervescence) and the mixture stored atroom temperature for one hour with occasional swirling. The solvent wasstripped off at reduced pressure. The crystalline residue Was trituratedwith cyclohexane and dried at reduced pressure. The infrared spectrum(Nujol) was consistent for the desired acid chloride.

Sodium 7-(3-benzyl 1,2,4oxadiazole-5-one-4-acetamido)cephalosporanate.--A solution of3-benzyl-l,2,4- oxadiazole-5-one-4-acetyl chloride (prepared from 5.0g., 0.0214 mole of the acid) in 100 ml. of acetone was added all at onceto a rapidly stirred mixture of 5.72 g. (0.0214 mole) of7-aminocephalosporanic acid, 7.2 g. (0.0856 mole) of sodium bicarbonate,ml. of water and 50 ml. of acetone at room temperature. After stirringfor 45 minutes the reaction mixture was extracted twice with ethylacetate. The aqueous phase was layered with ethyl acetate and acidifiedwith 42% phosphoric acid. Insoluble material was removed by filtrationprior to separation of the phases. The aqueous phase was extracted twicemore with ethyl acetate. The combined ethyl acetate extracts were washedthree times with water, dried with sodium sulfate, filtered and treatedwith 7.6 ml. (0.02 mole) of sodium 2-ethylhexanoate in l-butanol. Afterabout onehalf hour the mixture was concentrated somewhat and the productcollected by filtrtaion, washed with ethyl acetate and dried in vacuoover phosphorus pentoxide; yield 6.4 g., decomposes 2l5220 with priordarkening above about The infrared and nuclear magnetic resonancespectra were consistent for the desired product.

Analysis.Calcd. for C H N O SNa: C, 49.41; H, 3.75; N, 10.98. Found: C,48.89; H, 3.86; N, 10.86.

7 [N (Phenylacetimidoyl)aminoacetamido)cephalosporanic acid.A solutionof 5.2 g. of sodium 7-(3-benzyl-1,2,4-oxadiazole-5-one 4acetamido)cephalosporanate in 200 ml. of water was hydrogenated in thepresence of commercial Raney nickel catalyst (No. 28) on a Parrhydrogenation apparatus at an initial pressure of 50 p.s.i. for one hourat room temperature. The catalyst was removed by filtration. Thefiltrate was adjusted to pH 3 with 6 N hydrochloric acid and then to pH4.2 with 20% aqueous sodium hydroxide. The solution was concentrated toa small volume. A small amount of solid was removed by filtration. Thefiltrate was concentrated to dryness. Ethyl acetate was added to theresidue and then distilled off to remove the last traces of water. Theethyl acetate treatment was repeated once; yield 3.2 g. The infrared andnuclear magnetic resonance spectra were consistent for the desiredproduct.

Example 2 5 Hydroxy-3-phenyl-l,2,4-oxadiazole.-A solution of 103.1 g. (1mole) of benzonitrile in 400 ml. of 95% ethanol was added to a mixtureof 53 g. (0.5 mole) of sodium carbonate, 69.5 g. (1 mole) ofhydroxylamine hydrochloride, 200 m1. of water and 300 ml. of 95%ethanol. The mixture was refluxed for 18 hours. Most of the ethanol wasdistilled off and additional water was added. The product, whichcrystallized on cooling, was filtered, washed with water, and air dried.The product was dissolved in benzene and the benzene distilled off toremove any remaining water leaving a crystalline residue of benzamideoxirne (lit. ref. Beil. 9, 304, II 214).

Ethyl chloroformate (96 ml., 1 mole) was added gradually from a droppingfunnnel to a stirred solution of benzamide oxirne in 250 ml. of warmbenzene plus 80.5 ml. (1 mole) of pyridine. The exothermic reactioncaused the reaction mixture to reflux. The mixture was refluxed for 15minutes longer, cooled somewhat, and 500 ml. of water added. The benzenewas distilled off. The aqueous mixture remaining was refluxed for 2.5hours. After cooling in an ice bath the crystalline product wascollected by filtration. The crude product was purified by dissolving indilute aqueous sodium hydroxide and acidifying with acetic acid; yield122.9 g., m.p. 196200 (lit. ref. Beil. 27, 644, II 698).

3 Phenyl 1,2,4 oxadiazole--one-4-acetic acid-A mixture of 32.2 g. (0.193mole) of ethyl bromoacetate, 10.5 g. of sodium methoxide, 28.5 g. (0.176mole) of 5- hydroxy-3-phenyl-1,2,4-oxadiazole and 300 ml. of methanolwas heated at reflux for 4 hours and then stored at room temperatureovernight. To the reaction mixture was added g. of sodium hydroxide and30 ml. of water. The solution was heated at reflux for 1.5 hours. Water(70 ml.) was added and the methanol distilled off at reduced pressure.Acidification of the aqueous concentrate with 6 N hydrochloric acid gavean oily crystalline precipitate which was filtered and air dried (solidA); yield 29 g. A second crop of product precipitated from the filtrate(solid B); yield 2.1 g. The infrared spectrum of solid B was consistentfor 3 phenyl-1,2,4-oxadiazole-5-one-4-acetic acid.

Solid A was recrystallized from 1:1 95 ethanol water with a carbontreatment giving 7.8 g. of 5-hydroxy-3-phenyl-1,2,4-oxadiazole as thefirst crop. The filtrate deposited a second crop (8.9 g.) (solid C)which had an infrared spectrum consistent for3-phenyl-1,2,4-oxadiazole-5-one- 4-acetic acid.

Solid C was recrystallized by dissolving in ethyl acetate and dilutingwith a large volume of Skellysolve B. The material which immediatelyseparated (solid D) was removed. The filtrate was stored in the cold toallow the product to crystallize; yield 3.8 g. The infrared and nuclearmagnetic resonance spectra were fully consistent for 3-phenyl-1,2,4-oxadiazole-5-one-4acetic acid.

Analysis.Calcd. for C H N O /2H O: C, 52.40; H, 3.96; N, 12.23; H O,3.93. Found: C, 52.37; H, 4.76; N, 12.50; H O, 3.26.

Solid D was purified with ethyl acetate-Skellysolve B as describedgiving additional product. The filtrate from solid C was concentrated todryness and the residue purified with ethyl acetate-Skellysolve B. Thusan additional 2.8 g. of 3-phenyl-1,2,4-oxadiazole-5-one-4-acetic acidwas obtained.

S-Phenyl 1,2,4 oxadiazole-5-one-4-acetyl chloride. A suspension of 1.0g. (0.00455 mole) of 3-phenyl-1,2,4- oxadiazole-5-one-4-acetic acid in50 ml. of methylene chloride was gassed briefly with hydrogen chlorideand 1.1 g. (0.00528 mole) of phosphorus pentachloride added. The mixturewas stored at room temperature for 2 hours. The solvent was distilledfrom the resulting clear solution leaving the acid chloride as an oil.

Potassium 7-(3-phenyl 1,2,4 oxadiazole 5 one-4-acetamido)-cephalosporanate.-A solution of 3 phenyl- 1,2,4 oxadiazole 5one-4-acetyl chloride prepared from 1 g. of the acid in 10 ml. ofacetone was added to a solution of 1.24 g. (0.00455 mole) of7-amino-cephalosporanic acid, 1.5 g. of sodium bicarbonate, 30 ml. ofwater and 20 ml. of acetone at room temperature with rapid stirring.After stirring for 0.5 hour the reaction mixture was diluted with 30 ml.of water and extracted once with ethyl acetate. The aqueous phase waslayered with ethyl acetate and acidified with 42% phosphoric acid. Twomore extractions with ethyl acetate were made. The combined ethylacetate extracts were washed three times with water, dried with sodiumsulfate, filtered and treated with 1.7 ml. (0.00455 mole) of potassium2-ethylhexanoate in l-butanol giving an oil which slowly crystallized.The mixture was concentrated slightly and fresh ethyl acetate added. Thecrystalline product was filtered, washed with ethyl acetate, dried invacuo over phosphorus pentoxide and further dried in vacuo at 60 for 3hours; yield 1.58 g., decomposes 145150 with prior darkening above aboutThe infrared and nuclear magnetic resonance spectra were consistent forthe desired product.

Analysis.-Calcd. for C2UH17N4OQSK'ZHZOI C, H, 3.86; N, 10.21. Found: c,44.15; H, 4.37; N, 10.71.

7 [N-(Benzimidoyl)aminoacetamido]cephalosporanic acid.A solution of 1.0g. of potassium 7-(3-phenyl-l,2,4- oxadiazole 5 one 4acetamido)cephalosporanate in 150 ml. of water was hydrogenated for 3hours in the presence of commercial Raney nickel catalyst (No. 28) on aParr hydrogenation apparatus at room temperature at an initial pressureof 50 psi. The catalyst was removed by filtration and washed well withwater. The filtrate was adjusted to pH 5 with 6 N hydrochloric acid,concentrated to a small volume, readjusted to pH 5, and concentrated todryness. The residue was triturated with anhydrous ether and acetone,filtered, and dried in vacuo over phosphorus pentoxide; yield 0.8 g.,decomposes -145 with prior darkening above about 130. The infrared andnuclear magnetic resonance spectra were consistent for the desiredproduct.

Example 3 3-(p-Chlorobenzyl) 1,2,4 oxadiazole 5 one.A solution of 151.6g. (1 mole) of p-chlorophenylacetonitrile in 200 ml. of 95 ethanol wasadded to a stirred mixture of 69.5 g. (1 mole) of hydroxylaminehydrochloride, 53 g. (0.5 mole) of sodium carbonate, 200 ml. of waterand 400 ml. of 95 ethanol. The mixture was heated at reflux for 19hours. Most of the ethanol was stripped off at reduced pressure causingthe crude p-chlorophenylacetamide oxirne to separate as an oil. Theproduct was extracted into 600 m1. of benzene. The benzene extract waswashed two times with water. The product which started to crystallizefrom the benzene phase was kept in solution by warming. The benzenesolution was concentrated to about 400 ml.

Ethyl chloroformate (108.5 g., 1 mole) was added dropwise to a stirredmixture of the above prepared solution of p-chlorophenylacetamide oxirnein benzene plus 80.5 ml. (1 mole) of pyridine. The reaction mixture wasbrought to reflux after about three-quarters of the ethyl chloroformatehad been added. The mixture was refluxed for 25 minutes after theaddition was completed. The mixture was cooled to room temperature and500 ml. of water were added. The benzene was distilled off. The aqueousresidue was heated at reflux for 2 hours. The reaction mixture wascooled in an ice bath and the aqueous phase decanted from the oil. Tothe residual oil was added a solution of 45 g. of sodium hydroxide in500 ml. of water and the mixture refluxed for 2 hours. The cooledmixture was adjusted to pH 3-4 with concentrated hydrochloric acid. Theproduct was collected by filtration and washed with water. The productwas dissolved in 500 ml. of boiling benzene, the hot solution filteredand the filtrate diluted with 500 ml. of cyclohexane giving 56 g. ofproduct. The crude product was recrystallized from 1:1 95 ethanol-waterwith a carbon treatment and then from benzene. The product was dissolvedin dilute aqueous sodium hydroxide and the filtered solution wasacidified with glacial acetic acid; m.p. 168- 172, yield, after dryingin vacuo over phosphorus pentoxide, 21.6 g. The infrared and nuclearmagnetic resonance 13 spectra were consistent for 3-(p-chlorobenzyl)1,2,4- oxadiazole-S-one.

Analysis.Calcd. for C H ClN O C, 51.32; H, 3.35; N, 13.30. Found: C,51.46; H, 3.63; N, 13.46, 13.53.

The combined benzene filtrates deposited a second crop of product whichwas purified by dissolving in dilute sodium hydroxide andreprecipitating with acetic acid; yield 3.2 g., m.p. l66-170.

3-(p-Chlorobenzyl) 1,2,4 oxadiazole one-4- acetic acid.A solution of23.2 g. (0.0992 mole) of 3-(pchlorobenzyl) 1,2,4 oxadiazole 5 one in 450ml. of methanol was stirred at room temperature with 20 g. of powderedMolecular Sieves (Linde 4A) for 1.5 hours. The Molecular Sieves wereremoved by filtration. The filtrate was added to a freshly preparedsolution of sodium methoxide in methanol made by adding 2.5 g. (0.11mole) of sodium to 100 ml. of methanol. Ethyl bromoacetate (18.4 g.,0.11 mole) was added. The stirred mixture was refluxed for 19 hours inan apparatus protected from atmospheric moisture. The solvent wasdistilled 01f at reduced pressure. To the residue was added 200 ml. ofglacial acetic acid and 100 ml. of 6 N hydrochloric acid and the mixtureheated at reflux for 2 hours. The volatile materials were stripped offat reduced pressure. Water and benzene Were added to the residue causingthe product to crystallize. The product was filtered, washed with waterand benzene and twice recrystallized from toluene with a carbontreatment each time; yield 8.9 g., mp. 145-149. The infrared and nuclearmagnetic resonance spectra were fully consistent for the desiredproduct.

Analysis.-Calcd. for C H ClN O C, 49.18; H, 3.38; N, 10.43. Found: C,49.43: H. 3.54: N. 10.68. 10.69.

Sodium 7 [3-(p-chlorobenzyl)-l,2,4-oxadiazole-5-one- 4-acetamido]cephalosporanate.-A suspension of 4.0 g. of 3 (p chlorobenzyl)1,2,4-oxadiazole-5-one-4-acetic acid in 200 ml. of methylene chloridewas gassed for about 1 minute with hydrogen chloride. Phosphoruspentachloride (0.04 g.) was added (effervescence) and the reactionmixture was stored at room temperature for 2 hours with occasionalswirling. The solvent was stripped off at reduced pressure. The residualoil was triturated with cyclohexane and dried in vacuo to remove thelast traces of solvent leaving 3 (p chlorobenzyl)-1,2,4-oxadiazole-5-one-4-actyl chloride as residue.

A solution of the acid chloride in 90 ml. of acetone was added in oneportion to a rapidly stirred mixture of 4.07 g. of7-aminocephalosporanic acid, 5.02 g. of sodium bicarbonate, 150 m1. ofwater and 60 ml. of acetone at room temperature. After stirring for 20minutes the mixture was extracted three times with ethyl acetate. Theaqueous phase was layered with ethyl acetate and acidified with 42%phosphoric acid. Two more extractions were made on the aqueous phasewith ethyl acetate. The solid which had separated on acidification wasremoved by filtration during the second extraction. The combined ethylacetate extracts were washed three times with water, dried with sodiumsulfate, filtered, treated with 5.6 ml. (0.0149 mole) of sodium2-ethylhexanoate in l-butanol and concentrated somewhat giving acrystalline precipitate. The precipitate was filtered, washed with ethylacetate and was concluded to be mostly the sodium salt of the side chainacid; yield 1.2 g. The filtrate was stripped of solvent at reducedpressure. The residue was triturated with anhydrous ether giving 3.3 g.of solid. The solid was extracted at room temperature with 150 ml. ofacetone. A small amount of insoluble material was filtered off. Theacetone filtrate was stripped of solvent. The residue was trituratedwith anhydrous ether, filtered and dried in vacuo over phosphoruspentoxide; yield 2.8 g., mp. 155-160 decomp. The infrared and nuclearmagnetic resonance spectra were consistent for sodium 7 [3 (pchlorobenzyl) 1,2,4- oxadiazole-5-one-4-acetamido] cephalosporanate.

7 [N (p-Chlorophenylacetimidoyl)aminoacetamido] cephalosporanic acid.Asolution of 2.6 g. of sodium 7- [3 (p chlorobenzyl) 1,2,4oxadiazole-5-one-4-acetamido1cephalosporanate in 200 ml. of water washydrogenated in the presence of commercial Raney nickel catalyst (No.28) on a Parr hydrogenation apparatus at an initial pressure of 50p.s.i. for 2 hours at room temperature. The catalyst was removed byfiltration. The filtrate was adjusted to pH 2.5 with 6 N hydrochloricacid and a small amount of solid filtered off. The filtrate was adjustedto pH 4 with 20% sodium hydroxide and concentrated to dryness. Ethylacetate was added to the residue and this distilled oif at reducedpressure to remove water. This was repeated with acetone. Water (15 ml.)was added to the residue. The resulting gummy partially crystallinesolid was collected by filtration and washed with water. The solid wastriturated with acetone, three times with 1:1 ethanol-acetone and washedwell with acetone giving a crystalline product. The product was dried invacuo over phosphorus pentoxide and further dried for 4 hours in avacuum oven at 60; yield 0.84 g., decomposes 1952l0 with darkening aboveabout The infrared and nuclear magnetic resonance spectra wereconsistent for 7 [N (p chlorophenylacetimidoyl)aminoacetamido]cephalosporanic acid.

Analysis.-Calcd. for C H ClN O S- /2H O; C, 49.03; H, 4.53 N, 11.44; HO, 1.84. Found: C, 49.15; H, 4.85; N, 11.52, 11.46; H O, 1.57.

Example 4 3 Benzyl 1,2,4 oxadiazole-5-one.Phenylacetonitrile (235 g., 2moles) was added to a stirred mixture of 167 g. (2.4 moles) ofhydroxylamine hydrochloride 127 g. (1.2 mole) of sodium carbonate, 1 l.of methanol and 600 ml. of water. The stirred mixture was heated at 50(solution) for 21 hours. The methanol was distilled off at reducedpressure. The crude amide oxime which separated as an oil was extractedinto 700 ml. of chloroform. The chloroform extract was washed 3 timeswith water, dried with sodium sulfate, filtered and about 200 ml. ofsolvent distilled olT at reduced pressure to remove any remaining waterleaving a dry solution of phenylacetamide oxime in chloroform.

Ethyl chloroformate ml., 2 moles) was added in a slow stream to astirred and cooled (2530) solution of the above obtained chloroformsolution of phenylacetamide oxime, an additional 1.5 1. of chloroformand 280 ml. (2 moles) of triethyl amine. The solution was concentratedat reduced pressure (in 3 batches) until a thick residue of product andtriethylamine hydrochloride remained. The batches were combined andextracted 3 times with water. The chloroform-product phase was strippedat reduced pressure to remove the remainder of the solvent. To theresidue was added a solution of 120 g. of sodium hydroxide in 1 l. ofwater. The vigorously stirred mixture was heated rapidly during about 10minutes to 70-80' causing most of the material to go into solution. Thecooled (25) mixture was extracted once with 300 ml. of benzene to removea small amount of insoluble oil. The ice cooled aqueous phase wasacidified (pH 2-3) with concentrated hydrochloric acid. The product wascollected by filtration, Washed with water and air dried; yield 153.1 g.The product was suspended in 800 ml. of water and aqueous sodiumhydroxide solution (69 g. of sodium hydroxide in 200 ml. of water) addedin slight excess of the amount needed to dissolve all of the solid. Thesolution was carbon treated, filtered and acidified with con centratedhydrochloric acid. The product was filtered, washed with water and airdried; yield 119.6 g., m.p. Ill-114.

3-Benzyl-1,2,4-oxadiazole-5-one-4-acetic acid.A solution of 10 g. (0.057mole) of 3-benzyl-1,2,4-oxadiazole- 5-one, 10.4 g. (0.0625 mole) ofethyl bromoacetate and 8.8 ml. (0.0625 mole) of triethylamine in 100 ml.of tetrahydrofuran was stirred at room temperature for 5 hours. Duringthis time a thick precipitate of triethylamine hydrobromide formed.After storage at room temperature for 64 hours the solid was removed byfiltration. The filtrate was concentrated at reduced pressure to removesolvent. The residue of crude ethyl 3-benzyl-1,2,4-oxadiazole one 4acetate was combined with 80 ml. of glacial acetic acid and 40 ml. of 6N hydrochloric acid and the solution refluxed for 1.5 hours. Thevolatile materials were distilled oil at reduced pressure. An infraredspectrum of the residue indicated that the hydrolysis of the ester wasincomplete. Therefore, 80 ml. of glacial acetic acid and 40 ml. of 6 Nhydrochloric acid were added and the solution was refluxed for 2.5hours. The volatile materials were removed at reduced pressure. Waterand benzene were added to the residue and the mixture stored at roomtemperature overnight to crystallize the product. After cooling in anice bath the product was filtered and air dried. Recrystallization fromtoluene with a carbon treatment gave 8.0 g. (60% yield) of 3- benzyl1,2,4 oxadiazole-5-one-4-acetic acid; mp. 113- 115.

7 (3 Benzyl 1,2,4-oxadiazole-5-one-4-acetamido)-desacetoxycephalosporanic acid:

COOH

A solution of 5.0 g. (0.0214 mole) of 3-benzyl-1,2,4-oxadiazole-5-one-4-acetic acid in 200 ml. of methylene chloride wasgassed briefly (about 2 minutes) with hydrogen chloride Phosphoruspentachloride (5.8 g., 0.0278 mole) was added (effervescence) and themixture stored at room temperature for 1.5 hours with occasionalswirling. The solvent was stripped off at reduced pressure. Thecrystalline residue of 3-benzyl-1,2,4-oxadiazole-5- one-4-acetylchloride was triturated with cyclohexane and dried at reduced pressure.A solution of the acid chloride in 90 ml. of acetone was added all atonce to a rapidly stirred solution of 4.58 g. (0.0214 mole) of7-aminodesacetoxycephalosporanic acid, 7.2 g. (0.0856 mole) of sodiumbicarbonate, 150 ml. of water and 60 ml. of acetone at room temperature.After 20 minutes the reaction mixture was extracted once with ethylacetate whereupon the sodium salt of the product crystallized from theaqueous phase. The aqueous phase together with the precipitate which hadformed was layered with ethyl acetate and acidified with 42% phosphoricacid. Three extractions using a total of ca. 800 ml. of ethyl acetatewere made. The combined ethyl acetate extracts were washed 3 times withwater, dried with sodium sulfate, filtered and treated with 8.1 ml.(0.0214 mole) of sodium Z-ethylhexanoate in l-butanol giving aprecipitate. To this mixture was added 7 ml. of water resulting in acrystalline solid. The mixture was concentrated to above 500 ml. and thecrystalline sodium7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)desacetoxycephalosporanatecollected by filtration, washed with acetone and air dried; yield 7.6 g.The filtrate was concentrated at reduced pressure. The oily crystallineresidue was triturated with anhydrous ether giving 0.8 g. of sodiumsalt.

Both crops of sodium salt were combined (8.4 g.) and dissolved in 150ml. of water by slight warming. The solution was cooled in an ice bath,acidified with 42% phosphoric acid (causing the free acid toprecipitate) and 150 ml. of acetone added giving a solution. Thesolution was concentrated somewhat on a flash evaporator until the freeacid started to crystallize. After 2 hours at 0 the product,7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)desacetoxycephalosporanic acid, was filtered, washed with a small amountof water, air dried, and dried in vacuo over phosphorus pentoxide; yield4.31 g., decomposes 208 -210 with prior darkening above about 200. Theinfrared and nuclear magnetic resonance spectra were consistent i9? thedesired compound.

1 6 Example 5 7 [N (Phenylacetimidoyl)aminoacetamido]desace.

toxycephalosporanic acid.

-N\ CHI OOH A suspension of 4.16 g. of 7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido) desacetoxycephalosporanic acid in ml. of water wasadjusted to pH 7 with solid sodium bicarbonate causing most of thematerial to dissolve. Dioxane (50 ml.) was added and the slightly turbidsolution 'was hydrogenated in the presence of commercial Raney nickelcatalyst (No. 28) on a Parr hydrogenation apparatus at room temperatureat an initial pressure of 50 p.s.i. for 1 hour. The catalyst was removedby filtration. The filtrate was adjusted to pH 4.3 with 6 N hydrochloricacid, concentrated to about one half of its initial volume, againadjusted to pH 4.3, and further concentrated to a small volume causingthe product to precipitate. The product was filtered, washed with waterand acetone, dried in vacuo over phosphorus pentoxide and further driedin a vacuum oven at 60 for 4 hours; yield 1.96 g., decomposes 184-186with prior darkening above about The infrared and nuclear magneticresonance spectra were consistent for the desired compound.

Example 6 CONHz CONH: CH CONH; l t l t /N on. ooNn,

lTL

to produce, respectively, the compounds of the formulae 17 Whennecessary the amide is prepared by converting the corresponding acid toits acid chloride with thionyl chloride and thence to the amide bytreatment of the acid chloride with ammonia in the usual manner.

Example 7 The procedures of Example 2 are repeated while replacing thebenzonitrile used therein with an equimolar weight of the nitrile of theformula R-CN wherein R is dichloromethyl, trichloromethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl, respectively to produce,respectively, the corresponding compounds of the formulae 7-[N(Phenylacetimidoyl) aminoacetamido1cephalosporanic acid.- A solution of3-benzyl-1,2,4-oxadiazole- 5-one-4-acetyl chloride (prepared from 5.0g., 0.0214 mole of the acid) in 90 ml. of acetone was added in oneportion to a rapidly stirred mixture of 5.72 g. (0.0214 mole) of7-aminocephalosporanic acid, 7.2 g. (0.0856 mole) of sodium bicarbonate,150 ml. of water and 60 ml. of acetone at room temperature. Afterstirring for 45 minutes the reaction mixture was extracted twice withethyl acetate, the ethyl acetate extracts being discarded. The aqueousphase was layered with ethyl acetate and acidified with 42% phosphoricacid. Two more extractions with ethyl acetate were made. A quantity ofSOlld separated during the second extraction and was removed byfiltration. The combined ethyl acetate extracts were washed three timeswith water, dried with sodium sulfate, filtered, and treated with 8.1ml. (0.0214 mole) or sodium Z-ethylhexanoate in l-butanol giving acrystalline precipitate. The mixture was concentrated somewhat to removeany remaining water. The product, sodlum 7-(3benzyl-1,2,4-oxadiazole-5-one-4-acetamido)cephalosporanate, wascollected by filtration and washed with ethyl acetate (designated solidA). The filtrate was stripped of solvent and the residue triturated withanhydrous ether giving a filterable solid (designated solid B); yield0.2 g. Solid A was extracted by stirring with 500ml. of acetone. Theinsoluble portion (designated solid C, 7.1 g.) was removed byfiltration. The filtrate was stripped of solvent and the residuetriturated with anhydrous ether giving a filterable solid (designatedsolid D); yield 0.2 g. Solids B and D were concluded from their infraredspectra to be good quality sodium 7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)cephalosporanate.

A solution of solid C in water was layered with ethyl acetate andacidified with 42% phosphoric acid. Two more extractions of the aqueousphase were made with ethyl acetate. The combined organic extracts wereWashed three times with water, dried with sodium sulfate, filtered andthe solvent stripped oil at reduced pressure. To the residue was added500 ml. of water and the mixture stirred for about one hour in a waterbath at 40 causing the product,7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)cephalosporanic acid, tocrystallize; yield after air drying, 4.2 g. The infrared spectrum wasconsistent for good quality material.

A suspension of 4.2 g. of 7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)cephalosporanic acid, 0.2 g. of solid B and 0.2 g. ofsolid D in 200 ml. of water was brought into solution by addition ofsolid sodium bicarbonate to pH 6.5 The solution was hydrogenated in thepresence of commercial Raney nickel catalyst (No. 28) on a Parrhydrogenation apparatus at an initial pressure of 50 psi. at roomtemperature for two hours. During this time the theoretical amountv ofhydrogen was taken up. The catalyst was removed by filtration. Thefiltrate was adjusted to pH 4.5 with 6 N hydrochloric acid andconcentrated to dryness. To the residue was added ethyl acetate and thisstripped out to remove any remaining water. The process was repeatedonce using acetone in place of ethyl acetate giving a solid residue. Theresidue was extracted with ml. of warm ethanol the insoluble materialbeing removed by filtration. The filtrate was stored in the coldovernight and filtered to remove a small amount of solid. The filtratewas concentrated to a volume of about 50 ml. and diluted quickly with 50ml. of acetone. The precipitate which immediately separated was filteredoff. The filtrate was stored for about one hour at room temperaturewhile the product separated. The product, 7-[N-(phenylacetimidoyl)aminoacetamido]cephalosporanic acid, was filtered,washed with acetone, dried in vacuo over phosphorus pentoxide andfurther dried for 4 hours in a vacuum oven at 60; yield 1.34 g.,decomposes 168-170" with prior darkening above about The infrared andnuclear magnetic resonance spectra were consistent for the desiredproduct.

Example 9 Potassium 7-(3-phenyl1,2,4-oxadiazole-5-one-4-acetamido)desacetoxycephalosporanate.Asuspension of 1.5 g. of 3-phenyl-1,2,4-oxadiazole-5-one-4-acetic acid in60 ml. of methylene chloride was gassed with hydrogen chloride brieflyand 1.66 g. of phosphorus pentachloride added. The mixture was stirredat room temperature for 2 hours. The solvent was removed at reducedpressure from the solution leaving 3-phenyl-1,2,4-oxadiazole-5-one-4-acetyl chloride as an oil.

A solution of 3-phenyl-1,2,4-oxadiazole-5-one-4-acetyl chloride in asmall amount of acetone was added slowly with stirring to a mixture of1.45 g. of 7-amino-desacetoxycephalosporanic acid, 1.14 g. of sodiumbicarbonate, 40 ml. of water and 40 ml. of acetone while maintaining pH7-8 with sodium bicarbonate. The mixture was stirred for 0.5 hour whilebeing maintained at about pH 7.5 with hydrochloric acid. The reactionmixture was diluted with 40 ml. of water and extracted with ethylacetate. The aqueous phase was acidified with 42% phosphoric acid andextracted three times with ethyl acetate. The combined organic extractswere washed three times with water, dried with sodium sulfate andtreated with 2.5 ml. (0.0068 mole) of potassium 2-ethylhexanoate inl-butanol giving a crystalline product. The product was filtered, washedwith ethyl acetate and dried; yield 2.45 g., decomposes -185 Theinfrared and nuclear magnetic resonance spectra were consistent forpotassium 7-(3- phenyl 1,2, 4-oxadiazole-S-one-4-acetamido)desacetoxycephalosporanate.

Analysis.Calcd. for C H N O SK-2H O: C, 44.07; H, 3.90; N, 11.42. Found:C, 43.73; H, 3.84; N, 11.32.

Example 10 7 N- Benzimidoyl aminoacetamido] desacetoxycephalosporanicacid.-A solution of 2.1 g. of potassium 7- (3-phenyl1,2,4-oxadiazole-5-one-4-acetamido) desacetoxycephalosporanate in 200ml. of water was hydrogenated in the presence of commercial Raney nickelcatalyst on a Parr hydrogenation apparatus at room temperature at aninitial pressure of 50 p.s.i. for two hours. The catalyst was removed byfiltration. The filtrate was adjusted to pH 4.8 with 6 N hydrochloricacid and concentrated to dryness at reduced pressure. The residue wastriturated with anhydrous ether producing a filterable solid; yield 1.6g. The material was crystallized by dissolving in water and then'addingacetone to the cloud point; yield after drying in vacuo over phosphoruspentoxide, 0.39 g., decomposes 183-187. The infrared and nuclearmagnetic resonance spectra were consistent for the desired product.

Example 11 7 (3 Benzyl 1,2,4-oxadiazole--one-4-acetamido)-3-(l-pyridylmethyl)-3-cepheme-4-carboxylic acid betaine. A mixture of 8 g.of 7-(3-benZyl-l,2,4-oxadiazole-5-one-4- acetamido)cephalosporanic acid,32 g. of potassium thiocyanate, 8 ml. of water and 4 ml. of pyridine washeated at 6065 for 5 hours. To the reaction mixture which had beenstored at room temperature overnight was added 100 m1. of water. Theresulting solution was slowly acidified to pH 2 with 6 N hydrochloricacid with ice cooling. The aqueous phase was decanted from the gummyprecipitate. The precipitate was triturated three times with water andthe resulting solid, 7-(3-benzyl-1,2,4-oxadiazole- 5 one4-acetamido)-3-(l-pyridylmethyl)-3-cepheme-4- carboxylic acidthiocyanate was collected by filtration. The solid was suspended in 150ml. of water plus 40 ml. of a 25% solution of Amberlite LA-l resinacetate form in toluene [Spencer et al., J. Med. Chem. 9, 746 (1966),footnote 14] and the mixture stirred for one hour. The toluene-resinphase was separated and 40 ml. of a 25% solution of Amberlite LA-l resinacetate form in methyl isobutyl ketone was added and the mixture stirredfor one hour. The resulting emulsion was extracted six times withanhydrous ether. The aqueous phase was filtered and stored in therefrigerator overnight. The water was stripped off at reduced pressure.Ethyl acetate was added to the residue and this stripped out to removeany remaining water. The ethyl acetate treatment was repeated once. Thesolid was triturated with anhydrous ether and filtered giving 2.2 g. of7-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido) -3-,( l-pyridylmethyl-3-cepheme-4-carboxylic acid betaine; melts 130135, dec. 135145.

Am'berlite LA-1 resin is a mixture of secondary amines wherein eachsecondary amine has the formula wherein each of R R and R is analiphatic hydrocarbon radical and wherein R R and R contain in theaggregate from 11 to 14 carbon atoms; this particular mixture ofsecondary amines, which is sometimes referred to as Liquid Amine MixtureNo. I, is a clear amber liquid having the following physicalcharacteristics: viscosity at 25 C. of 70 cps.; specific gravity at C.of 0.845; refractive index at C. of 1.467; distillation range at 10 mm.:up to 160 C.-74%, above 220 C.17%.

Example 12 7 [N-(Phenylacetimidoyl)aminoacetamido]-3-(l-pyridylmethyl)-3-cepheme-4-carboxylic acid chloridehydrochloride.A solution of 2.1 g. of 7-(3-benzyl-1,2,4-oxadiazole 5one-4-acetamido)-3-(1-pyridylmethyl)-3-cepheme-4-carboxylic acid betainein 150 ml. of water and 50 ml. of dioxane plus 0.5 ml. of glacial aceticacid was hydrogenated in the presence of commercial Raney nickelcatalyst (No. 28) on a Parr hydrogenation apparatus at an initialpressure of 50 p.s.i. at room temperature for 1.5 hours. The catalystwas removed by filtration. The filtrate was concentrated to dryness atreduced presure, i.e. stripped. Ethyl acetate was added to the residueand stripped out to remove any remaining water. The ethyl acetateprocedure was repeated once. To the residue was added 30 ml. of waterand 2 ml. of 6 N hydrochloric acid and the resulting solution strippedto dryness at reduced pressure. The residue was dissolved in 20 ml. ofwater and again stripped to dryness. To the residue was added ethyl 20acetate and this stripped out to remove remaining water. The treatmentwith ethyl acetate was repeated once. The residue was triturated withacetone giving after filtration and drying in vacuo over phosphoruspentoxide 1.2 g. of product; gradually decomposes above about 140.

Example 13 3-(2-Thenyl)-1,2,4-oxadiazole-5-one.A mixture of 148 g. ofdistilled 2-thiopheneacet0nitrile, 97.3 g. of hydroxylaminehydrochloride, 74.1 g. of sodium carbonate, 600 ml. of methanol and 360ml. of water was heated at 50 for 19.5 hours. The methanol was strippedoff at reduced pressure. The product, 2-thiopheneacetamide oxime,separated as an oil. The oil was extracted into 600 ml. of chloroform.The chloroform extract was washed three times with water, dried oversodium sulfate and concentrated to a volume of about 500 ml.

Ethyl chloroformate (114.4 ml.) was added from a dropping funnel to asolution of the above obtained 2-thiopheneacetamide oxime in chloroformplus an additional 500 ml. of chloroform and 168 ml. of triethylaminewith cooling to keep the temperature from rising above 25. After theaddition of ethyl chloroformate was complete the solvent was distilledoff at reduced pressure. The residue was extracted with water to removetriethylamine hydrochloride. The organic phase was further stripped atreduced pressure to remove all solvent leaving an oil as residue. To theresidue was added a solution of 72 g. of

sodium hydroxide in 600 ml. of water and the mixture heated to 75 during15 minutes. The solution was cooled to room temperature and twiceextracted with benzene. The aqueous phase was carbon treated andacidified with cooling to pH 3.5 causing the product to crystallize. Theproduct was filtered, washed with water and air dried; yield 108.9 g.The product was purified by dissolving in aqueous sodium hydroxide (24g. of sodium hydroxide in 500 ml. of water), carbon treating thesolution and acidifying with concentrated hydrochloric acid. There wasobtained 84.3 g. of 3-(2-thenyl)-1,2,4-oxadiazole-5-one, m.p. 9697.5.

3- Z-Thenyl l ,2,4-oxadiazole-5-one-4-acetic acid.--A stirred mixture of18.2 g. of 3-(2-thenyl)-1,2,4-oxadiazole-5-one, 28 ml. of triethylamine,16.7 g. of bromoacetic acid and 300 ml. of tetrahydrofuran was heated at50 overnight. The precipitate of triethylamine hydrobrornide was removedby filtration. The filtrate was stripped at reduced pressure to removesolvent. Water (100 ml.) and sufiicient 20 %sodium hydroxide were addedto the residue to give a solution. The solution was acidified withconcentrated hydrochloric acid causing the product to separate as anoil. The mixture was layered with benzene and concentrated to a volumeof about 50 ml. at reduced pressure. The concentrate was extracted twicewith ethyl acetate. The combined ethyl acetate extracts were Washed withwater, dried over sodium sulfate, filtered and the filtrate treated with31 ml. (0.081 mole) of sodium 2-ethylhexanoate in l-butanol causingsodium 3-(2-thenyl)-l,2,4-oxadiazole-5-one-4-acetate to crystallize. Theproduct was filtered and washed well with ethyl acetate; yield 7.0' g.The infrared spectrum was consistent for the desired product.Acidification of an aqueous solution of the sodium salt produced thecrystalline free acid, 3-(2-thenyl)-1,2,4-oxadiazole-5-one-4-aceticacid.

Sodium 7-[3-(2-thenyl)-1,2,4-oxadiazole 5 one 4-acetamido]cephalosporanate.-A solution of 3-(2-thenyl)1,2,4-oxadiazole-5-one-4-acetic acid prepared from 6.5 g. of the sidiumsalt in ml. of methylene chloride was gassed for a few minutes withanhydrous hydrogen chloride, 6.7 g. of phosphorous pentachloride wasadded (eifervescence) and the mixture stirred at room temperature for 25minutes. The solvent was distilled off at reduced pressure. The residuewas triturated by decantation with cyclohexane and finally the solventwas completely removed at reduced pressure giving 3-(2-thenyl)-1,2,4-oxadiazole-5-one-4-acetyl chloride.

A solution of the acid chloride in 25 ml. of methylene chloride wasadded to a stirred solution of 7.4 g. of 7- aminocephalosporanic acidand 8.7 ml. if triethylamine at room temperature. The reaction mixturewas stirred at room temperature for one hour. The methylene chloride wasstripped olf at reduced pressure and the residue was dissolved in 200m1. of water. The acidified (42% phosphoric acid) aqueous silution wasextracted three times with ethyl acetate. The combined ethyl acetateextracts were washed twice with water, dried with sodium sulfate,filtered, concentrated somewhat to remove any remaining water andtreated with 9.4 ml. (1 equiv.) of sodium 2- ethylhexanoate inl-butanol. The crystalline product was collected by filtration andstored in a desiccator (solid A). The filtrate was stripped of solventat reduced pressure and the residue triturated with anhydrous ether. Theproduct was filtered and washed with anhydrous ether (solid B); yield5.7 g. The infrared and nuclear magnetic resonance spectra of solid Bwere consistent for the product, sodium 7[3-(2-thenyl)-1,2,4-oxadiazole-5-one-4- acetamido1- cephalosporanate.Solid A was concluded to be a mixture of product and the sodium salt ofthe side chain acid. A suspension of solid A in ethyl acetate-acetonewas stirred for 5-10 minutes and the insoluble portion filtered oif. Thefiltrate was concentrated to a small volume, a small amount ofprecipitate was removed by filtration and the filtrate was concentratedto dryness. The residue was triturated with anhydrous ether giving anadditional 1.32 g. of good quality product.

7 [N-(Z-thienylacetimidoyl)aminoacetamido]-cephalosporanic acid. Sodium7-[3-(2-thenyl)-1,2,4-0xadiazole 5-one-4-acetamido]cephalosporanate (6.8g.) was combined with 200 ml. of water and the system adjusted to pH 7.6with sodium bicarbonate giving a slightly hazy solution. The filteredsolution was adjusted to pH 7.3 with 6 N hydrochloric acid andhydrogenated in the presence of Raney nickel catalyst at an initialpressure of 50 p.s.i. at room temperature on a Parr hydrogenationapparatus for 1.75 hours. The catalyst was removed by filtration. Thefiltrate was adjusted to pH 2.7 with 6 N hydrochloric acid and a smallamount of insolube material filtered off. The filtrate was adjusted topH 4.2 with sodium hydroxide and stripped to dryness at reducedpressure. The residue was treated several times with ethyl acetate andthis stripped out to remove all water. The residue was dissolved in 80ml. of methanol, diluted with 80 ml. of acetone and filtered twice. Thefiltrate after storage in the cold overnight was filtered and strippedto dryness. The residue was triturated with acetone producing afilterable solid; yield 1.53 g. The solid was extracted with 1:1 95%ethanol-acetone. The insoluble portion was removed by filtration. Thefiltrate was diluted with acetone causing the product to precipitate.The product was collected by filtration and washed with acetone. Theinsoluble portion was again extracted with 1:1 ethanolacetone and theextract worked up in the same way giving a second crop of product; totalyield of both crops 0.17 g. The infrared and nuclear magnetic resonancespectra were consistent for the desired product.

The filtrates from both crops of product were combined, stripped todryness and the residue triturated with anhydrous ether to give anadditiinal 0.27 g. of product of somewhat poorer quality.

7 (3 Benzyl-1,2,4-oxadiazole-5-one-4-acetamido)-3- (2methyl-1,3,4-thiadiazol-5-ylthio)methylceph-3-em-4- carboxylic acid andsodium salt.A solution of sodium 22 7(3-benzyl-l,2,4-oxadiazole-5-one-4-acetamido)cephalosporanate in pH 6.4phosphate butter and an equivalent amount of2-mercapto-5-methyl-1,3,4-thiadiazole is stirred at 60 for 4 to 6 hours.The reaction mixture is acidified to pH 2 with 6 N hydrochloric acid andextracted with ethyl acetate. The organic phase is washed with water,dried with sodium sulfate, filtered, and evaporated in vacuo to give thefree acid.

The sodium salt is prepared by treating an ethyl acetate solution of thefree acid with an equivalent amount of sodium 2-ethylhexanoate inl-butanol.

Example 15 (JOOH 7 [N (Phenylacetimidoyl)aminoacetamido] 3-(2- methyl1,3,4-thiadiazol S-ylthio)methylceph-3-em-4- carboxylic acid.A solutionof 7-(3-benzyl-1,2,4-oxadiazole5-one-4-acetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthio)methylceph-3-em-4-carboxylic acid in dioxane is hydrogenated inthe presence of Raney nickel catalyst at an initial pressure of 50p.s.i. on a Parr hydrogenation apparatus at room temperature for 2hours. The catalyst is removed by filtration. The filtrate is evaporatedat reduced pressdre to give the product.

Example 16 i s crIi- NH-cHicNH N-N PHH 0 CH -s- I on \f 2 \S/ 3 I OOOH 7[N (Phenylacetimidoyl)aminoacetamido] 3-(2- methyl1,3,4-thiadiazol-5-ylthio)methylceph-3-em-4-carboxylic acid. A solutionof sodium 7-(3-benzyl-1,2,4- oxadiazole 5-one4-acetamido)-3-(2-methyl-1,3,4-thiadiazol-S-ylthio)methylceph-3-em-4-carboxylate in aqueous dioxane is hydrogenatedin the presence of Raney nickel catalyst at an initial pressure of 50p.s.i. on a Parr hydrogenation apparatus at room temperature for 2hours. The catalyst is removed by filtration. The filtrate is adjustedto pH 4.5 and evaporated to a small volume giving the product.

Example 17 3-(3-Thenyl)-1,2,4-oxadiazole-5-one.A mixture of 155.6 g. of3-thiopheneacetonitrile, 105.08 g. of hydroxylamine hydrochloride, 80.12g. of sodium carbonate, 6C0 ml. of methanol and 360 ml. of water washeated at 50 for 18 hours. The methanol was stripped off at reducedpressure. The product, 3-thiopheneacetamide oxime, separated as an oil.The oil was extracted into 600 ml. of chloroform. The chloroform extractwas washed three times with water and dried over sodium sulfate causingthe oxime to crystallize. The product was removed from the sulfate bydissolving it with 600 ml. of tetrahydrofuran and filtering off thesodium sulfate. The solvent was removed fnom the filtrate at reducedpressure. The crystalline residue was dissolved in 1000 ml. ofchloroform containing 176.4 ml. of triethylamine.

Ethyl chloroformate (120.1 ml.) was added from a dropping funnel to theabove obtained solution of 3-thiopheneacetamide oxime with cooling tokeep the temperature from rising above 25. After the addition of ethylchloroformate was complete the solvent was distilled off at reducedpressure. The residue was extracted with water to remove triethylam'inehydrochloride. The organic phase was further stripped at reducedpressure to remove all solvent leaving an oil as residue. To the residuewas added a solution of 75.6 g. of sodium hydroxide in 600 ml. of waterand the mixture heated to 75 during minutes. The solution was cooled toroom temperature and twice extracted with benzene. The aqueous phase wascarbon treated and acidified with cooling to pH 3.5 causing the productto crystallize. The product was collected by filtration, washed withwater and air dried. The product was purified by recrystallization frombenzene; yield 51.0 g. The product was further purified by dissolving inaqueous sodium hydroxide (11.24 g. of sodium hydroxide in 300 ml. ofwater) and acidifying with concentrated hydrochloric acid. There wasobtained 47.5 g. of 3-('3- thenyl)-1,2,4-oxadiazole-5-one, m.p. 63-64.5.

Sodium 3 (3 Thenyl) 1,2,4-oxadiazole-5-one-4-acetate.-A stirred, drymixture of 47.4 g. of 3-(3-thenyl)- 1,2,4-oxadiazole-5-one, 72.8 ml. oftriethylamine, 43.4 g. of brom oacetic acid and 750 ml. oftetrahydrofuran was heated at 50 overnight. The precipitate oftriethylamine hydrobromide was removed by filtration. The filtrate wasstripped at reduced pressure to remove solvent. Water (300 ml.) andsufficient sodium hydroxide were added to the residue to give asolution. The solution was carbon treated, cooled and acidified withconcentrated hydrochloric acid causing the acid form of the product toseparate as an oil. The mixture was extracted twice with ethyl acetate.The combined ethyl acetate extracts were washed with water, dried oversodium sulfate, filtered and the filtrate treated with 79.0 ml. (0.080mole) of sodium 2-ethylhexanoate in l-butanol causing sodium 3(3-thenyl)-1,2,4-oxadiazole-5-one-4-acetate to crystallize. The productwas collected by filtration and washed well with ethyl acetate; yield13.8 g. The infrared spectrum was consistent for the desired product.

Sodium 7-[3-(3-thenyl)-1,2,4-0xadiazole-5-one-4acetamido]cephalosporanate.A solution of 3 (3-thenyl)-1,2,4-oxadiazole-5-one-4-acetic acid prepared from 6.5 g. of the sodiumsalt in 85 ml. of methylene chloride was gassed for a few minutes withanhydrous hydrogen chloride. Then 6.7 g. of phosphorus pentachloride wasadded (elfervescence) and the mixture stirred at room temperature forminutes. The solvent was distilled off at reduced pressure. The residuewas triturated by decantation with cyclohexane and finally the solventwas completely removed at reduced pressure giving 3-(3-thenyl)-1,2,4-oxadiazole-5-one-4-acetyl chloride.

A solution of the acid chloride in 25 ml. of methylene chloride wasadded to a stirred solution of 7.4 g. of 7-aminocephalosporanic acid and8.7 ml. of triethylamine at room temperature. The reaction mixture wasstirred at room temperature for one hour. The methylene chloride wasstripped off at reduced pressure and the residue was dissolved in 200ml. of Water. The acidified (42% phosphoric acid) aqueous solution wasextracted 3 times with ethyl acetate. The combined ethyl acetateextracts were washed twice with water, dried with sodium sulfate,filtered, concentrated somewhat to remove any remaining water andtreated with 9.4 ml. (1 eqv.) of sodium 2-ethylhexanoate in l-butanol.The product separated as a gum. Decantation of the solvent andtrituration of the residue with fresh ethyl acetate produced solidproduct. The product was collected by filtration and washed well withethyl acetate (solid A); yield 9.4 g. The decanted solvent and washingswere combined and stripped at reduced pressure. The residue wastriturated with anhydrous ether. The product was collected by filtrationand washed with anhydrous ether (solid B); yield 2.7 g. The infraredspectra of solids A and B indicated them to be mixtures of product andthe sodium salt of the side chain acid. Each was suspended in acetoneand stirred for 5-10 minutes. The insoluble portions were filtered offand the filtrates concentrated to dryness. The residues were trituratedwith anhydrous ether giving solids C and D; yield 7.6 g. and 2.2 g.respectively. The infrared spectra indi- 24 cated both samples to begood quality product as the sodium salt.

7 [N (3-thienylacetimdoyl)aminoacetamido]-cepha losporanic acid.Sodium 7[3-(3-thenyl)-1,2,4-oxadiazole-5-one-4-acetamido]cephalosphoranate (5.0g.) was combined with 200 ml. of water and the system adjusted to pH 7.8with sodium bicarbonate giving a clear solution. The solution washydrogenated in the presence of Raney nickel catalyst at an initialpressure of 50 p.s.-i. at room temperature on a Parr hydrogenationapparatus for 1.5 hours. The catalyst was removed by filtration. Thelfiltrate was adjusted to pH 4.2 with 6 N hydrochloric acid and someinsoluble material separated. After concentration to a small volume atreduced pressure the material was collected by filtration (solid A);yield 1.4 g. The filtrate was stripped to dryness at reduced pressure.The residue was treated several times with ethyl acetate and thisstripped out to remove all water. The residue was dissolved in 30 ml.methanol, filtered to remove insoluble sodium chloride, diluted with 30ml. acetone filtered again and stored in the refrigerator overnight. Theproduct which had crystallized was collected by filtration and washedsparingly with acetone (solid B); yield 0.96 g.; its infrared andnuclear magnetic resonance spectra were consistent for the desiredproduct. Solid A was concluded to be a mixture of product and unreducedstarting material.

What is claimed is:

1. A compound of the formula wherein A is hydrogen, acetoxy,2-(5-methyl-1,3,4-thiadiazolyl)-thio, pyridinium, picolinium orlutidinium; and M is hydrogen, a pharmaceutically acceptable, nontoxiccation or an anionic charge when A is one of said quaternary ammoniumradicals; wherein Z is (lower)alkyl,

R1 @(CIIzM-u Wanton Ra Ba 0 \S wherein R is hydrogen, hydroxy, amino,methyl, methoxy or chloro.

5. A compound of claim 1 having the formula wherein Z is phenyl, benzyl,p-chlorobenzyl, 2-thenyl or thenyl.

6. A compound of claim 1 having the formula 10. The compound having theformula 30011 (JOOH wherein Z is phenyl, benzyl, p-chlorobenzyl,2-thenyl or 11. The compound having the formula 3-thenyl. O 7. Acompound of claim 1 having the formula II -0H,-| -0H,-c O J l 9 whereinZ is phenyl, benzyl, p-chlorobenzyl, Z-thenyl or COO 3 1 12. Thecompound havmg the formula 8. A compound of claim 1 having the formulaE!) -cH,-'N-om-c- O NN Nl zNCH i NHCH(|3H on, 0

N O=-N JCH -S JCH: N N O s NH(l3H(|3fi (3H l L $00K 0=o--N C o-om-s on,wherein Z is phenyl, benzyl, p-chlorobenzyl, Z-thenyl or $0011 3-thenyl.

9. The compound having the formula References cued UNITED STATES PATENTSo s 3,365,449 1/1965 Takano et a1. 260-243 C ]T L I fl, 3,687,949 8/1972Holdrege 260243 c N O=C N C-CHrWC-Clh NICHOLAS S. RIZZO, PrimaryExaminer US. Cl. X.R. 5003 424246

